MiSeq genome Sequencing

[wchathura]

Is it possible to sequence a genome around 400 mb using MiSeq sequencer??

[nucacidhunter]

Assuming 20M useful output reads (2x300 cycle), you will have 12Gb of data which is equivalent to 30x coverage. That most likely will not be enough to de novo assemble a whole genome, but certainly would provide coverage for large region of genome in various sizes of contigs.

[wchathura]

Thank you very much for the reply. however if there is a reference genome available for the organism is it possible to assemble the genome with 30x coverage??

[GenoMax]

If a reference genome is available then you are likely going to "align" instead of assemble. There is possibility of "reference guided" assembly if the reference genome available is not for the exact species you are sequencing.

[wchathura]

Thank you very much for your reply. exact reference genome is available for the species we are trying to sequence. however the thing is that Miseq manual says that it can be used to sequence small genomes and they are not specifying the size of the the genome that can be efficiently sequenced with it. so i was wondering whether it could be used to sequence a genome of about 400 mb s ??

[GenoMax]

What you actually sequence on MiSeq is 300-500 bp fragments that ultimately come from your source DNA (that can be of any size). These are generated during the library preparation. What you should think about is what kind of coverage you want achieve (30 x is probably enough in your case for re-sequencing). This can vary depending on what you are looking to get from the data (e.g. very rare variants may need deeper coverage).

[wchathura]

Thanks again for the quick reply. now it is clear to me.